Acidic Endochitinase Research Articles

Legume-bacteria-soil interaction networks linked to improved plant productivity and soil fertility in intercropping systems

Intercropping with legumes is an effective strategy to increase plant productivity, reduce nitrogen (N) inputs, and maintain soil fertility in an industrial monoculture forest. However, the facilitation/complementarity mechanism of the interactions between legumes (keystone species in intercropping), bacteria and soil are still unknown, and the contribution of this triple interaction to plant growth and ecosystem function is still difficult to predict. Taking the introduction of Dalbergia odorifera (D. odorifera), into eucalyptus plantations as an example, we evaluated the changes in nutrient uptake, phytohormone contents, N metabolism-related enzyme activities, and root-associated bacteria of D. odorifera under different N applications (0, 3, 6 and 12 g pot−1) and different planting patterns (monoculture and intercropping), and investigated the regulatory mechanism of these cascaded changes on productivity and soil fertility in response to intercropping. The results showed that intercropping increased the total biomass by 5.90–56.29% compared to monoculture; in particular, intercropping in combination with 6 g N pot−1 had superiority in promoting the gibberellin content, glutamine synthetase activity, nitrogenase activity, soil fertility, and bacterial diversity, as well as the relative abundance of bacteria with the potential to undergo aerobic chemoheterotrophy, N fixation and N respiration. The differentially expressed genes identified via transcriptome analysis were mainly involved in metabolic pathways, signal transduction, and the tricarboxylic acid cycle. To integrate the associations between plant traits, bacterial communities, and soil properties, we built a multivariable network based on multiple plant traits, soil properties, differentially accumulated genera, and differentially expressed genes in monoculture and intercropping. These results led to the identification of candidates related to intercropping advantages, including two genes (acidic endo chitinase, Na+/K+ transporter) and four bacterial genera (Bacteroides, Coxiella, Pseudomonas, SJA-15). More specifically, moderate fertilization of eucalyptus/D. odorifera intercropping systems create a more stable interaction network with additional module hubs. These results highlight the importance of bacterial diversity and the plant-bacteria-soil interaction network in maintaining soil fertility and plant productivity, which may help fertilization practices in eucalyptus plantations and provide guidance for the ecological sustainability of introducing D. odorifera into industrial forests.

Genomic regions associated with herbicide tolerance in a worldwide faba bean (Vicia faba L.) collection

Weeds represent one of the major constraints for faba bean crop. The identification of molecular markers associated with key genes imparting tolerance to herbicides can facilitate and fasten the efficient and effective development of herbicide tolerant cultivars. We phenotyped 140 faba bean genotypes in three open field experiments at two locations in Lebanon and Morocco against three herbicide treatments (T1 metribuzin 250 g ai/ha; T2 imazethapyr 75 g ai/ha; T3 untreated) and one in greenhouse where T1 and T3 were applied. The same set was genotyped using genotyping by sequencing (GBS) which yield 10,794 high quality single nucleotide polymorphisms (SNPs). ADMIXTURE software was used to infer the population structure which revealed two ancestral subpopulations. To identify SNPs associated with phenological and yield related traits under herbicide treatments, Single-trait (ST) and Multi-trait (MT) Genome Wide Association Studies (GWAS) were fitted using GEMMA software, showing 10 and 14 highly significant associations, respectively. Genomic sequences containing herbicide tolerance associated SNPs were aligned against the NCBI database using BLASTX tool using default parameters to annotate candidate genes underlying the causal variants. SNPs from acidic endochitinase, LRR receptor-like serine/threonine-protein kinase RCH1, probable serine/threonine-protein kinase NAK, malate dehydrogenase, photosystem I core protein PsaA and MYB-related protein P-like were significantly associated with herbicide tolerance traits.

Proteome Responses to Individual Pathogens and Abiotic Conditions in Rice Seedlings.

Rice plants under field conditions experience various biotic and abiotic stresses and are adapted to survive using a molecular cross-talk of genes and their protein products based on the severity of a given stress. Seedlings of cultivated variety ASD16 (resistant to fungal disease, blast; tolerant to abiotic stress, salinity) were subjected to salt, drought, high temperature and low temperature stress as well as infection by Rhizoctonia solani and Xanthomonas oryzae pv. oryzae (causing reemerging diseases such as sheath blight and leaf blight), respectively, the sheath blight and bacterial leaf blight pathogens. Leaf proteome was analyzed using two-dimensional electrophoresis and differentially expressed proteins were identified using mass spectrometry. In addition to many other differentially expressed proteins, acidic endochitinase was found to be upregulated during fungal infection and drought treatment, and a germin-like protein upregulated during fungal infection and high temperature stress. These two proteins were further validated at the gene expression level using reverse transcription-PCR in dual stress experiments. Pot culture plants were subjected to fungal infection followed by drought and drought followed by fungal infection to validate chitinase gene expression. Similarly, plants subjected to fungal infections followed by high temperature stress and vice versa were used to validate the expression of germin-like protein-coding gene. The results of the present study indicate that chitinase and germin-like protein are potential targets for further exploration to develop rice plants resistant or tolerant to biotic and abiotic stresses.

Proteomics and post-secretory content adjustment of Nicotiana tabacum nectar.

The tobacco nectar proteome mainly consists of pathogenesis-related proteins with two glycoproteins. Expression of nectarins was non-synchronous, and not nectary specific. After secretion, tobacco nectar changed from sucrose rich to hexose rich. Floral nectar proteins (nectarins) play important roles in inhibiting microbial growth in nectar, and probably also tailoring nectar chemistry before or after secretion; however, very few plant species have had their nectar proteomes thoroughly investigated. Nectarins from Nicotiana tabacum (NT) were separated using two-dimensional gel electrophoresis and then analysed using mass spectrometry. Seven nectarins were identified: acidic endochitinase, β-xylosidase, α-galactosidase, α-amylase, G-type lectin S-receptor-like serine/threonine-protein kinase, pathogenesis-related protein 5, and early nodulin-like protein 2. An eighth nectarin, a glycoprotein with unknown function, was identified following isolation from NT nectar using a Qproteome total glycoprotein kit, separation by SDS-PAGE, and identification by mass spectrometry. Expression of all identified nectarins, plus four invertase genes, was analysed by qRT PCR; none of these genes had nectary-specific expression, and none had synchronous expression. The total content of sucrose, hexoses, proteins, phenolics, and hydrogen peroxide were determined at different time intervals in secreted nectar, both within the nectar tube (in vivo) and following extraction from it during incubation at 30°C for up to 40h in plastic tubes (in vitro). After secretion, the ratio of hexose to sucrose substantially increased for in vivo nectar, but no sugar composition changes were detected in vitro. This implies that sucrose hydrolysis in vivo might be done by fixed apoplastic invertase. Both protein and hydrogen peroxide levels declined in vitro but not in vivo, implying that some factors other than nectarins act to maintain their levels in the flower, after secretion.

Proteomic analysis by iTRAQ-PRM provides integrated insight into mechanisms of resistance in pepper to Bemisia tabaci (Gennadius)

BackgroundThe Bemisia tabaci is a major leaf feeding insect pest to pepper (Capsicum annuum), causing serious damage to pepper growth and yield. It is particularly important to study the mechanism of pepper resistance to B. tabaci, and to breed and promote the varieties of pepper resistant to B. tabaci. However, very limited molecular mechanism is available about how plants perceive and defend themselves from the destructive pest. Proteome technologies have provided an idea method for studying plant physiological processes in response to B. tabaci.ResultsHere, a highly resistant genotype and a highly susceptible genotype were exposed to B. tabaci feeding for 48 h to explore the defense mechanisms of pepper resistance to B. tabaci. The proteomic differences between both genotypes were compared using isobaric tag for relative and absolute quantification (iTRAQ). The quantitative data were validated by parallel reaction monitoring (PRM). The results showed that 37 differential abundance proteins (DAPs) were identified in the RG (resistant genotype), while 17 DAPs were identified in the SG (susceptible genotype) at 48 h after B. tabaci feeding. 77 DAPs were identified when comparing RG with SG without feeding. The DAP functions were determined for the classification of the pathways, mainly involved in redox regulation, stress response, protein metabolism, lipid metabolism and carbon metabolism. Some candidate DAPs are closely related to B. tabaci resistance such as annexin D4-like (ANN4), calreticulin-3 (CRT3), heme-binding protein 2-like (HBP1), acidic endochitinase pcht28-like (PR3) and lipoxygenase 2 (LOX2).ConclusionsTaken together, this study indicates complex resistance-related events in B. tabaci interaction, provides novel insights into the molecular mechanism underlying the response of plant to B. tabaci, and identifies some candidate proteins against B. tabaci attack.

Computational modeling and functional characterization of a GgChi: A class III chitinase from corms of Gladiolus grandiflorus

The present study describes the predicted model and functional characterization of an endochitinase (30 kDa) from corms of Gladiolus grandiflorus. ESI-QTOF-MS generated peptide showed 96% sequence homology with family 18, Class III acidic endochitinase of Gladiolus gandavensis. Purified G. grandiflorus chitinase (GgChi) hydrolyzed 4-methylumbelliferyl β-d-N,N′,N′′-triacetylchitotriose substrate showing specific endochitinase activity. Since no structural details of GgChi were available in the Protein Data Bank (PDB), a homology model was predicted using the coordinate information of Crocus vernus chitinase (PDB ID: 3SIM). Ramachandran plot indicated 84.5% in most favored region, 14.8% in additional and 0.6% in generously allowed region while no residue in disallowed region. The predicted structure indicated a highly conserved (β/α)8 (TIM barrel) structure similar to the family 18, class III chitinases. The GgChi also showed sequence and structural homologies with other active chitinases. The GgChi (50 μg/disc) showed no antibacterial activity, but did provide mild growth inhibition of phytopathogenic fungus Fusarium oxysporum at a concentration of 500 μg/well Similarly, insect toxicity bioassays of GgChi (50 μg) against nymphs of Bemisia tabaci showed 14% reduction in adult emergence and 14% increase in mortality rate in comparison to control values. The GgChi (1.5 mg) protein showed significant reduction in a population of flour beetle (Tribolium castaneum) after 35 days, but lower reactivity against rice weevil (Sitophilus oryzae). The results of this study provide detai.led insight on functional characterization of a family 18 class III acidic plant endochitinase.

Cloning and characterization of a chitinase from Thermobifida fusca reveals Tfu_0580 as a thermostable and acidic endochitinase

Being capable of hydrolyzing chitin, chitinases have various applications such as production of N-acetylchitooligosaccharides (COSs) and N-acetylglucosamine (GlcNAc), degrading chitin as a consolidated bioprocessing, and bio-control of fungal phytopathogens. Here, a putative chitinase in Thermobifida fusca, Tfu_0580, is characterized. Tfu_0580 was purified by homogeneity with a molecular weight of 44.9 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Tfu_0580 displayed a clear activity against colloidal chitin, which is comparable to a commercial Streptomyces griseus chitinase. Enzyme activities against p-nitrophenyl β-D-N,N′,N′′-triacetylchitotriose (p-NP-(GlcNAc)3), N,N′-diacetyl-β-D-chitobioside (p-NP-(GlcNAc)2) and p-nitrophenyl N-acetyl-β-D-glucosaminide (p-NP-(GlcNAc)) showed that Tfu_0580 exhibited highest activity against p-NP-(GlcNAc)3. Further optimization of the enzyme activity conditions showed: 1) an optimum catalytic activity at pH 6.0 and 30 °C; 2) activity over broad pH (4.8–7.5) and temperature (20–55 °C); 3) stimulation of activity by the metallic ions Ca2+ and Mn2+.

Effects of As stress on contents of saponin and flavonoid, key enzymes activities of Panax notoginseng and its proteomic analysis

Field plot experiments were conducted to study the effect of two-year consecutive As stress [As(V): 0, 20, 80, 140, 200 and 260 mg·kg-1] on contents of As, saponin and flanovoids, the enzyme activities of phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), and squalene synthase (SS) in main root, fibrous root and rhizome and shoot, and proteome of three-year old Panax notoginseng in Wenshan prefecture, Yunnan Province, China. The results showed that total saponin content of fibrous root decreased with increase in As treatment concentration. Total saponin contents of shoot and rhizome increased with 140 mg·kg-1 As treatment compared with control. SS activity of rhizome was higher than that of shoot. Flavonoid contents of diffe-rent plant parts decreased with increase in As treatment concentration. With 140 mg·kg-1 As treatment, activities of PAL and CHS in rhizome were higher than that in shoot. CHS activities in shoot and rhizome were lower, and PAL activities were higher than those of the control. As contents in different plant parts of P. notoginseng increased with increase in As treatment concentration. The highest As content was observed in fibrous root. With 140 mg·kg-1 As treatment, twenty-one diffe-rential proteins (ratio >2, P<0.05) were identified in the inoculated compared to the control. The down-regulated proteins included phosphoribulokinase, heat shock protein, NAD(P)-binding rossmann-fold superfamily proteinisoform, monodehydroascorbate reductase and cytochrome b6-f complex iron-sulfur subunit. The up-regulated proteins included CDC27 family protein, acidic endochitinase isoform, symbiosis receptor-like kinase precursor, isoflavone reductase-like protein, phospho-2-dehydro-3-deoxyheptonate aldolase, putative protein kinase superfamily protein, malate dehydrogenase, glyoxalase I isoform and glutamine synthetase cytosolic isozyme. In general, with two-year consecutive As stress, As contents in different plant parts of P. notoginseng increased, which not only affected the photosynthesis and energy, but also decreased the antioxidation and resilience, and induced the increased expression of protein involved in detoxication, resulted in decrease in the contents of flavonoid and saponin. The tolerant threshold value of P. notoginseng for As was 140 mg·kg-1.

Proteomic identification of gravitropic response genes in peanut gynophores

Peanut (Arachis hypogaea L.) is one of the most important oil-bearing crops in the world. The gravitropic response of peanut gynophores plays an essential role in peanut reproductive development. In this study, we developed an in vitro culture system and applied it to the study of peanut gynophore gravitropism. By comparing the proteomes of gynophores grown in vitro with the tip pointing upward (gravity stimulation sample) and downward (natural growth control) at 6h and 12h, we observed 42 and 39 with significantly altered expression pattern at 6 and 12h, respectively. Out of these proteins, 13 proteins showed same expression profiling at both 6h and 12h. They were identified by MALDI-TOF/TOF and further characterized with quantitative real time RT-PCR. Among the 13 identified proteins, two were identified as class III acidic endochitinases, two were identified as Kunitz trypsin protease inhibitors, and the remaining proteins were identified as pathogenesis-related class 10 protein, Ara h 8 allergen isoform 3, voltage-dependent anion channel, gamma carbonic anhydrase 1, germin-like protein subfamily 3 member 3 precursor, chloride channel, glycine-rich RNA-binding protein and gibberellin receptor GID1. Real time RT-PCR analysis revealed that transcriptional regulation is consistent with expression at the protein level for class III acidic endochitinase, Kunitz trypsin protease inhibitor, chloride channel and pathogenesis-related class 10 protein, while the expression of the other 7 proteins might be regulated at post-transcriptional levels. This study identified several potential gravitropic response proteins in peanut gynophores and helps to understand early gravitropic responses in peanut gynophores. The gravitropic response of the peanut gynophores plays an essential role in peanut production. However, the molecular mechanism responsible for gravitropic responses in the peanut gynophores has not been explored yet. The result generated in this study may provide in vitro culture system for gravitropism study of plant gravitropic response and novel insights into the proteome-level response and give a more comprehensive understanding of early gravitropic response in peanut gynophores. This article is part of a Special Issue entitled: Translational Plant Proteomics.

How can plant virus satellite RNAs alter the effects of plant virus infection? A study of the changes in the Nicotiana benthamiana proteome after infection by Peanut stunt virus in the presence or absence of its satellite RNA

Peanut stunt virus (PSV), which belongs to the Cucumovirus genus, is a pathogen of legumes. Certain PSV strains associated with a satellite RNA (satRNA) modify the symptoms of infected plants and interfere with plant metabolism. We used PSV-P genomic transcripts (GTs) with and without PSV-P satRNA and a comparative proteomic 2D-DIGE/MS study to assess their effects on Nicotiana benthamiana infection. When the proteomes of the PSV-P genomic transcripts-infected (no satRNA present) and mock-inoculated plants were compared 29 differentially regulated proteins were found. When comparisons were made for plants infected with PSV-P-GT in the presence or absence of satRNA, and for mock-infected plants and those infected with the satRNA-associated PSV-P-GT, 40 and 60 such proteins, respectively, were found. The presence of satRNA mostly decreased the amounts of the affected host proteins. Proteins involved in photosynthesis and carbohydrate metabolism, for example ferredoxin-NADP-reductase and malate dehydrogenase, are among the identified affected proteins in all comparisons. Proteins involved in protein synthesis and degradation were also affected. Such proteins include chaperonin 60β--whose abundance of the proteins changed for all comparisons--and aminopeptidase that is a satRNA- or PSV-P-GT/satRNA-responsive protein. Additionally, the levels of the stress-related proteins superoxide dismutase and acidic endochitinase Q increased in the PSV-P-GT- and PSV-P-GT/satRNA-infected plants. This study appears to be the first report on plant proteome changes in response to a satRNA presence during viral infection and, as such, may provide a reference for future studies concerning the influence of satRNAs during viral infections.

Caterpillar Labial Saliva Alters Tomato Plant Gene Expression

We examined the effects of Helicoverpa zea caterpillar labial saliva on tomato plant gene expression. Caterpillars with labial salivary glands (mock-ablated) and without (ablated) were fed on tomato plants for 24 hr; then, the leaf mRNA was analyzed with tomato microarrays. Analysis of the transcript profiles revealed 384 expressed sequence tags (ESTs) that were significantly altered due to herbivory compared to the non-wounded plants. The majority of the ESTs were quantitatively altered more so by mock-ablated caterpillars with labial salivary glands than ablated caterpillars. Particularly notable, ESTs encoding acid phosphatase, arginase, acidic endochitinase, dehydrin, polyphenol oxidase, protease inhibitors, and threonine deaminase were more highly stimulated by mock-ablated caterpillars than ablated caterpillars. In addition, tomato leaves were mechanically wounded with scissors and painted with labial salivary gland extract, autoclaved salivary gland extract, or water, and compared to non-wounded tomato plants. After 4 hr, these leaves were collected and a tomato microarray analysis of the mRNA revealed correlation of the gene expression of these leaves altered by mechanical wounding and painted with salivary gland extract to the gene expression of leaves fed on by mock-ablated caterpillars. We show that caterpillar labial saliva is an important component of herbivory that can alter plant gene expression.

Composition, Vigor, and Proteome of Mature Soybean Seeds Developed under High Temperature

The effects of high temperature treatment on soybean [Glycine max (L.) Merr.] seed composition, vigor, and proteome were investigated using mature dry seeds harvested from plants grown in environment‐controlled chambers. High day/night temperatures (37/30°C) from stages R5 through R8 altered ratios of individual fatty acids to total fatty acid compared to the control (27/18°C). Concentration of sugars decreased, but total protein and phytic acid concentration were unchanged. High temperature resulted in a greater proportion of abnormal seeds, but normal‐appearing seed exhibited reduced germination and vigor. Proteomic analysis detected 20 protein identities whose accumulations were changed by the high temperature. Fourteen spots were identified as seven subunits of seed storage proteins. The remaining six proteins were identified as those responding to abiotic stresses or having a function in respiration: (i) sucrose binding protein, (ii) Class III acidic endochitinase, (iii) heat shock protein (HSP22), (iv) late embryo abundant protein, (v) Bowman–Birk proteinase inhibitor, and (vi) formate dehydrogenase. High temperature during seed development changed soybean seed composition and decreased seed vigor, but also changed seed protein expression profiles.

Transgenic tomato plants expressing a Lycopersicon chilense chitinase gene demonstrate improved resistance to Verticillium dahliae race 2.

An acidic endochitinase gene (pcht28) isolated from Lycopersicon chilense was introduced into tomato (L. esculentum) through Agrobacterium-mediated transformation, using the CAMV 35S promoter. Transgenic plants demonstrated a high level of constitutive expression of pcht28 and chitinase enzyme activity. Kanamycin-resistant R1 plants (resulting from self-pollination of transgenic plants) as well as R2 plants were evaluated for their tolerance to Verticillium dahliae (race 1 and 2 for R1 plants and race 2 for R2 plants) in the greenhouse. They demonstrated a significantly (P<0.05) higher level of tolerance to the fungi compared to the nontransgenic plants, as measured by foliar disease symptoms, vascular discoloration, and vascular discoloration index. The transgenic plants produced in this study represent a source of genetic resistance to Verticillium dahliae.

Spatial distribution of chitinases and β‐1,3‐glucanase transcripts in bean arbuscular mycorrhizal roots under low and high soil phosphate conditions

Arbuscular mycorrhizal (AM) fungi extensively colonize the root cortex under low‐soil‐phosphate (P) conditions, whereas infection is limited under high‐P conditions. Fungal growth under both P conditions might be influenced by plant defence‐related gene expression. In this study, we used in situ hybridization methods to compare the cellular localization of three defence‐related mRNAs in non‐infected bean roots and in relation to fungal infection units. In non‐infected and infected roots, mRNAs encoding acidic and basic endochitinases were generally most abundant in the vascular cylinder. High‐P‐grown mycorrhizal roots showed localized accumulation of the acidic endochitinase mRNA in cortical cells containing arbuscules and in their immediate vicinity (one to five cell layers). The pattern of accumulation of the basic endochitinase mRNA was not affected by P or AM fungal infection. At the low P concentration, the β‐1,3‐glucanase mRNA accumulated predominantly in the vascular cylinder of non‐infected roots. Suppression of β‐1,3‐glucanase mRNA accumulation in these tissues was observed in non‐infected roots at the high‐P and in mycorrhizal roots at both P concentrations. The observed suppression extends at least several mm from fungal infection units, characterizing a systemic effect. Beta‐1,3‐glucanase mRNA accumulated also around a number of cortical cells containing arbuscules only at the low P concentration. The localized accumulations of the endochitinase and β‐1,3‐glucanase mRNAs suggest that the encoded proteins might be involved in the control of intraradical fungal growth.

The secretory nature of the lesion of carrot cell variant ts11, rescuable by endochitinase.

The carrot cell variant ts11 is unable to form somatic embryos at the non-permissive temperature of 32 degrees C, but the block can be overcome by the addition of a 32-kDa acidic endochitinase to the medium. In this work we conducted a cyto-histological analysis of the blocked embryo forms. The morphology of the endomembrane system is altered; in particular, the ER is dilated and may show electron-dense precipitates and continuity with the plasma membrane. These morphological alterations do not occur in the presence of externally-added endochitinase. We also noticed modifications of the culture medium that are probably related to the morphological observations: the total amount of secreted proteins is reduced and pulse-chase experiments revealed that, compared with wild-type cells, the secretion of major polypeptides is reduced while new minor polypeptides are secreted. Western blot analysis revealed the presence of the binding protein BiP, a resident of the ER and of glutamine synthase, a cytosolic protein, in the medium of ts11 but not wild-type cells. These results indicate that ts11 is altered in the secretory pathway but do not clarify the role of endochitinase.

Differential expression of defense-related genes in arbuscular mycorrhiza

Differential expression of plant defense related genes may contribute to the control of arbuscular mycorrhiza growth and development. The expression of chitinases, β-1, 3-glucanases, and genes involved in the biosynthesis of isoflavonoid phytoalexins has been studied in these symbiotic associations. Relative to noninfected roots, mycorrhizal roots showed a transient induction of endochitinase activities followed by suppression, under low and high P conditions. The suppression was attenuated under high P. Differential reductions in the steady-state level of mRNAs encoding a basic and an acidic endochitinase was observed. Suppression of β-1, 3-glucanase activities, two glucanase mRNAs, and chalcone isomerase mRNA levels was also observed. In situ localization of several defense-related mRNAs showed a predominant accumulation in the vascular cylinder of mycorrhizal and noninfected roots, under both P conditions. The levels of these mRNAs were systemically suppressed in arbuscular mycorrhizae (AM). Additionally, the accumulation of mRNAs encoding an acidic endochitinase and a β-1, 3-endoglucanase was enhanced in and around cells containing arbuscules, under high and low P conditions, respectively. The differential regulation of defense-related genes may contribute to the observed pattern of intraradical fungal growth. A hypothetical model for the functional roles and regulation of endochitinases and β-1, 3-endoglucanases in arbuscular mycorrhiza is proposed. Key words: arbuscular mycorrhiza, differential gene expression, plant defense genes, chitinases, β-1, 3-glucanases, phosphate.

Isolation of an osmotic stress- and abscisic acid-induced gene encoding an acidic endochitinase from Lycopersicon chilense.

We have identified one osmotic stress- and abscisic acid-responsive member of the endochitinase (EC 3.2.1.14) gene family from leaves of drought-stressed Lycopersicon chilense plants, a natural inhabitant of extremely arid regions in South America. The 966-bp full-length cDNA (designated pcht28) encodes an acidic chitinase precursor with an amino-terminal signal peptide. The mature protein is predicted to have 229 amino acid residues with a relative molecular mass of 24,943 and pI value of 6.2. Sequence analysis revealed that pcht28 has a high degree of homology with class II chitinases (EC 3.2.1.14) from tomato and tobacco. Expression of the pcht28 protein in Escherichia coli verified that it is indeed a chitinase. Northern blot analysis indicated that this gene has evolved a different pattern of expression from that of other family members reported thus far. It is highly induced by both osmotic stress and the plant hormone abscisic acid. Southern blot analysis of genomic DNA suggested that the pcht28-related genes may form a small multigene family in this species. The efficiency of induction of the gene by drought stress, in leaves and stems, is significantly higher in L. chilense than in the cultivated tomato. It is speculated that, besides its general defensive function, the pcht28-encoded chitinase may play a particular role in plant development or in protecting plants from pathogen attack during water stress.

Rhizobium Lipooligosaccharides Rescue a Carrot Somatic Embryo Mutant.

At a nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot cell mutant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type embryos. The causative component in the conditioned medium has previously been identified as a 32-kD acidic endochitinase. In search of a function for this enzyme in plant embryogenesis, several compounds that contain oligomers of N-acetylglucosamine were tested for their ability to promote ts11 embryo formation. Of these compounds, only the Rhizobium lipooligosaccharides or nodulation (Nod) factors were found to be effective in rescuing the formation of ts11 embryos. These results suggest that N-acetylglucosamine-containing lipooligosaccharides from bacterial origin can mimic the effect of the carrot endochitinase. This endochitinase may therefore be involved in the generation of plant analogs of the Rhizobium Nod factors.

A carrot somatic embryo mutant is rescued by chitinase.

At the nonpermissive temperature, somatic embryogenesis of the temperature-sensitive (ts) carrot cell mutant ts11 does not proceed beyond the globular stage. This developmental arrest can be lifted by the addition of proteins secreted by wild-type cells to the culture medium. From this mixture of secreted proteins, a 32-kD glycoprotein, designated extracellular protein 3 (EP3), that allows completion of somatic embryo development in ts11 at the nonpermissive temperature was purified. On the basis of peptide sequences and biochemical characterization, EP3 was identified as a glycosylated acidic endochitinase. The addition of the 32-kD endochitinase to ts11 embryo cultures at the nonpermissive temperature appeared to promote the formation of a correctly formed embryo protoderm. These results imply that a glycosylated acidic endochitinase has an important function in early plant somatic embryo development.

Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco.

Complementary DNA clones encoding two isoforms of the acidic endochitinase (chitinase, EC 3.2.1.14) from tobacco were isolated. Comparison of amino acid sequences deduced from the cDNA clones and the sequence of peptides derived from purified proteins show that these clones encode the pathogenesis-related proteins PR-P and PR-Q. The cDNA inserts were not homologous to either the bacterial form of chitinase or the form from cucumber but shared significant homology to the basic form of chitinase from tobacco and bean. The acidic isoforms of tobacco chitinase did not contain the amino-terminal, cysteine-rich "hevein" domain found in the basic isoforms, indicating that this domain, which binds chitin, is not essential for chitinolytic activity. The accumulation of mRNA for the pathogenesis-related proteins PR-1, PR-R, PR-P, and PR-Q in Xanthi.nc tobacco leaves following infection with tobacco mosaic virus was measured by primer extension. The results indicate that the induction of these proteins during the local necrotic lesion response to the virus is coordinated at the mRNA level.